Composition and Method for Treating Neuronal Ceroid Lipofuscinosis

ABSTRACT

Provided herein are methods for treatment of a neurodegenerative disease, such as neuronal ceroid lipofuscinosis including administering to a subject in need of such treatment a composition comprising a therapeutically effective amount of an agent that mediates upregulation of TPP 1.

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.61/734,679 filed Dec. 7, 2012, which is incorporated by reference hereinin its entirety.

SUPPORT

This invention was made with government support under contract numbersAT6681, NS64564 and NS71479 awarded by the National Institutes ofHealth. The government has certain rights in the invention.

TECHNICAL FIELD

The present invention relates to a composition and method for treating aneurodegenerative disease, such as neuronal ceroid lipofuscinosis.

BACKGROUND

Neuronal ceroid lipofuscinosis (NCL) is a group of neurodegenerativediseases mainly composed of typical autosomal recessive lysosomalstorage disorders. The NCLs can be characterized by clinicalmanifestations like progressive mental deterioration, cognitiveimpairment, visual failures, seizures and deteriorating motor functionaccompanied by histological findings such as the accumulation ofautofluorescent storage material in neurons or other cell types (1). TheNCLs have been subdivided into several groups (Type 1-10) based on theage of onset, ultrastructural variations in accumulated storagematerials, and genetic alterations unique to each specific disease type(2, 3).

Late infantile neuronal ceroid lipofuscinosis (Jansky-Bielschowskydisease, LINCL, Type 2) typically produces symptoms at the age of 2-4years, progresses rapidly and ends in death between ages 8 to 15 as aresult of a dramatic decrease in the number of neurons and other cells(2, 4). LINCL is associated with mutations in the Cln2 gene, a 13 exonand 12 intron gene of total length of 6.65 kb mapped to chromosome11p15.5. The Cln2 gene encodes lysosomal tripeptidyl tripeptidase I(TPP-I or pepstin insensitive protease), a 46 KD protein that functionin the acidic environment of the lysosomal compartment to removetripeptides from the amino terminus of proteins (5, 6). This mutation inthe Cln2 gene results in a deficiency and/or loss of function of theTPP1 protein that leads to intralysosomal accumulation ofautofluoroscent lipopigments known as ceroid-lipofuscin (5). Currentlythere is no established treatment or drugs available for this disease;all approaches are merely supportive or symptomatic, indicating a needfor novel therapeutic approaches (7). However, there are differentvariants of Cln2 mutations and there have been reports that residualTPP-I activity can be found in patients with LINCL, indicating thatthere must be a few copies of normal Cln2 gene remaining in patientsaffected with LINCL (8, 9).

SUMMARY

Provided herein is a method for treatment of a neurodegenerativedisease. The neurodegenerative disease may be neuronal ceroidlipofuscinosis, Alzheimer's disease, Huntington's disease, Amyotrophiclateral sclerosis (ALS), Parkinson's disease, including Parkinson's plusdiseases such as multiple system atrophy (MSA), progressive supranuclearpalsy (PSP), corticobasal degeneration (CBD) or dementia with Lewybodies (DLB). The neurodegenerative disorder may be characterized bydefective autophage. Such disorders include Alzheimer's, Parkinson'sdisease, and Huntington's disease. The neurodegenerative disease may bea lysosomal storage disorder. The lysosomal storage disorder may be, forexample, Tay-Sach's disease, Fabry disease, Niemann-Pick disease,Gaucher disease, Hunter Syndrome, Alpha-mannosidosis,Aspartylglucosaminuria, Cholesteryl ester storage disease, ChronicHexosaminidase A Deficiency, Cystinosis, Danon disease, Farber disease,Fucosidosis, or Galactosialidosis.

The method may comprise administering to a subject in need of suchtreatment a composition comprising a therapeutically effective amount ofan agent that mediates upregulation of TPP1. The agent may be alipid-lowering drug. The lipid-lowering drug may be a fibrate. Thefibrate may be gemfibrozil or fenofibrate. The neuronal ceroidlipofuscinosis may be late infantile neuronal ceroid lipofuscinosis orBatten disease. TPP1 may be upregulated by increasing TPP1 mRNA levels,increasing TPP1 protein levels, increasing TPP1 enzymatic activity, oractivating a PPARα-RXRα heterodimer.

The composition may further comprise a therapeutically effective amountof all-trans retinoic acid. The agent may be a fibrate. Administeringall-trans retinoic acid and the fibrate may provide a greatertherapeutic effect in the subject than administration of all-transretinoic acid or the fibrate alone.

Also provided herein is a method for treatment of neuronal ceroidlipofuscinosis, comprising administering to a subject in need of suchtreatment a composition comprising a therapeutically effective amount ofan agent, wherein the agent restores TPP1 activity. The agent may be afibrate. The fibrate may be gemfibrozil or fenofibrate. Thetherapeutically effective amount of the fibrate may be lower when thefibrate is administered in combination with all-trans retinoic acid.

Further provided herein is a method for treatment of neuronal ceroidlipofuscinosis, comprising administering to a subject in need of suchtreatment a composition comprising a therapeutically effective amount ofan agent that mediates upregulation of a gene selected from the group ofCln1, Cln2, Cln3, and any combination thereof. The agent may be afibrate. The fibrate may be gemfibrozil or fenofibrate.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows gemfibrozil and fenofibrate upregulating TPP1 mRNA andfunctionally active protein in mouse brain cells. (A, B) Mouse primaryastrocytes were treated with 25 μM gemfibrozil in serum-free DMEM/F12for 2, 6, 12, and 24 hrs followed by monitoring the mRNA expression ofCln1, Cln2 and Cln3 by semi-quantitative RT-PCR (A) and qPCR (B) (forCln2). (C, D) Mouse astrocytes were treated with differentconcentrations of gemfibrozil for 24 hrs under the same cultureconditions followed by monitoring the mRNA expression of Cln2 bysemi-quantitative RT-PCR (C) and real-time PCR (D). (E) Mouse primaryastrocytes were treated with 25 μM gemfibrozil and 10 μM fenofibrate for24 hrs under the same culture conditions followed by Western blot forTPP1. (F) Densitometric analysis of TPP1 expression (relative toβ-Actin) by gemfibrozil and fenofibrate treatment. (G) Mouse primaryastrocytes were treated with different concentrations of gemfibrozil andfenofibrate under similar culture conditions and were double-labeled forTPP1 (red) and GFAP (green). Scale bar=10 μM. (H, J, L) Mouse primaryneurons were isolated from different parts of the brain and were treatedwith 25 μM gemfibrozil and 10 μM fenofibrate in neurobasal mediacontaining B27-AO for 24 hrs and were double-labeled for TPP1 (red) andβ-tubulin (green) (H—for cortical neurons, J—for hippocampal neurons,L—for striatal neurons). DAPI was used to stain nuclei. Scale bar=20 μM.(I, K, M) Mouse neurons were treated with 25 μM gemfibrozil under sameculture conditions for 24 hrs followed by Western blot for TPP1 (I—forcortical neurons, K—for hippocampal neurons, M—for striatal neurons).Graphs represent the densitometric analysis of TPP1 level (relative toβ-Actin). (N) Mouse primary neurons were treated with differentconcentrations of gemfibrozil and fenofibrate in B27-AO containingNeurobasal media for 24 hrs followed by the activity assay using cellextract containing 5 μg of total protein. (O) Mouse primary astrocyteswere treated with different concentrations of gemfibrozil andfenofibrate in serum free DMEM/F12 medium for 24 hrs followed by theactivity assay using cell extract containing 5 μg of total protein. Allresults are mean±SEM of at least three independent experiments. p*<0.05vs control; p**<0.01 vs control.

FIG. 2 shows gemfibrozil and fenofibrate upregulating TPP1 mRNA andprotein in human primary astrocytes and SHSY5Y neuronal cells. (A, B)Human primary astrocytes were treated with 25 μM gemfibrozil inserum-free DMEM/F12 for 2, 6, 12 and 24 hrs followed by monitoring themRNA expression of Cln2 by semi-quantitative RT-PCR (A) and real-timePCR (B). (C-F) Human primary astrocytes were treated with differentconcentrations of gemfibrozil and fenofibrate for 12 hrs under the sameculture conditions followed by monitoring the mRNA expression of Cln2 bysemi-quantitative RT-PCR (C—for gemfibrozil, E—for fenofibrate) andreal-time PCR (D—for gemfibrozil, F—for fenofibrate). (G) Human primaryastrocytes were treated with 25 μM of gemfibrozil and 10 μM fenofibratefor 24 hrs under similar culture conditions and were double-labeled forTPP1 (red) and GFAP (green). (H) SH-SY5Y cells were treated with 25 μMof gemfibrozil and 10 μM fenofibrate in B27-AO containing Neurobasalmedia for 24 hrs and were double-labeled for TPP1 (red) and β-tubulin(green). DAPI was used to stain nuclei. All results are mean±SEM of atleast three independent experiments. p*<0.05 vs control. UN-Untreated.Scale bar=10 μM.

FIG. 3 shows involvement of PPARα in fibrate drug-mediated upregulationof TPP1 mRNA and protein. (A-E) Mouse primary astrocytes isolated fromPPARα^(−/−) and PPARβ^(−/−) and wild type mouse were treated withdifferent concentrations of gemfibrozil and fenofibrate in serum freeDMEM/F12 for 24 hrs followed by monitoring the mRNA expression of Cln2by semi-quantitative RT-PCR (A, C) and real-time PCR (B, D) and proteinlevel of TPP1 by Western blot (E). (F) Densitometric analysis of TPP1levels (relative to β-Actin) in PPARα^(−/−) and PPARβ^(−/−) and wildtype astrocytes by gemfibrozil and fenofibrate treatment. p^(a)<0.05 vsWT control; p^(b)<0.05 vs PPARβ^(−/−) control; ns—not significant w.r.tPPARα^(−/−) control (G, H) Mouse primary astrocytes isolated from WTmice were pre-treated with GW9662 for 30 min followed by treatment with25 μM gemfibrozil under similar culture conditions. The mRNA expressionof Cln2 was monitored by semi-quantitative RT-PCR (G) real time PCR (H).p^(c)<0.05 vs control; p^(d)<0.05 vs only GW treated. (I-K) Mouseprimary astrocytes isolated from PPARα^(−/−) and PPARβ^(−/−) and WT micewere treated with 25 μM gemfibrozil and 10 μM fenofibrate in serum freeDMEM/F12 for 24 hrs and double-labeled for TPP1 (red) and GFAP (green)(I—for WT, J—for PPARα^(−/−) and K—for PPARβ^(−/−) astrocytes). DAPI wasused to stain nuclei. UN—No treatment. Scale bar=10 μM. (L-N) Mouseprimary astrocytes isolated from PPARα^(−/−) and PPARβ^(−/−) and WT micewere treated with 25 μM gemfibrozil and 10 μM fenofibrate in serum freeDMEM/F12 for 24 hrs. Whole cell extracts containing 5 μg total proteinwas incubated at 37° C. with 250 μM 7-amido-4-methylcoumarin in 96 wellplates and readings were taken at an interval of 30, 45, 60, 90 and 120mins. Mean values were taken and plotted in graphical format (L—for Wildtype cells, M—for PPARα^(−/−) cells and N—for PPARβ^(−/−) cells). Allresults are mean±SEM of at least three independent experiments.

FIG. 4 shows oral administration of gemfibrozil upregulates TPP1 in vivoin cortical and nigral astrocytes and neurons of WT and PPARβ^(−/−), butnot PPARα^(−/−) mice. WT, PPARα^(−/−) and PPARβ^(−/−) mice (n=4 in eachgroup) were treated with 7.5 mg/kg body wt/day of gemfibrozil (dissolvedin 0.1% methylcellulose) or vehicle (0.1% methylcellulose) via gavage.After 21 d of treatment, mice were killed and cortical and nigralsections were double labeled for TPP1 (red) along with either GFAP(green) or NeuN (green) or TH (green). DAPI was used to visualizenucleus. (A1-A3, B1-B3) TPP1 levels were compared between the astrocytesand neurons of the cortical sections of vehicle treated and gemfibroziltreated (A1, B1) WT, (A2, B2) PPARα^(−/−), & (A3, B3) PPARβ^(−/−) mice.(A1-A3: GFAP and TPP1; B1-B3: NeuN and TPP1). Scale bar=20 μM. (A4, B4)Higher magnification images showing co-localization of TPP1 and GFAP inthe (A4) cortical astroglia and co-localization of NeuN and TPP1 in (B4)cortical neurons of gemfibrozil treated mice (WT and PPARβ^(−/−)). Scalebar=10 μM. (C1-C3, D1-D3) TPP1 levels were compared between theastrocytes and neurons of the nigral sections of vehicle treated andgemfibrozil treated (C1, D1) WT, (C2, D2) PPARα^(−/−) & (C3, D3)PPARβ^(−/−) mice. Scale bar=20 μM. (C1-C3: GFAP and TPP1; D1-D3: TH andTPP1). (C4, D4) Higher magnification images showing co-localization ofTPP1 and GFAP in the (C4) nigral astroglia and co-localization of TH andTPP1 in (D4) nigral TH neurons of gemfibrozil treated mice (WT andPPARβ^(−/−)). Scale bar=10 μM. All results represent analysis of each ofthree cortical and nigral sections of each of four different mice pergroup.

FIG. 5 shows oral administration of gemfibrozil upregulates TPP1 in vivoin the hippocampus of WT and PPARβ^(−/−), but not PPARα^(−/−) mice. WT,PPARα^(−/−) and PPARβ^(−/−) mice (n=4 in each group) were treated with7.5 mg/kg body wt/day of gemfibrozil (dissolved in 0.1% methylcellulose)or vehicle (0.1% methylcellulose) via gavage. After 21 d of treatment,mice were killed and hippocampal (CA1 and dentate gyrus) sections weredouble labeled for TPP1 (red) and GFAP (green). DAPI was used tovisualize nucleus. (A-C) TPP1 levels were compared between the dentategyrus region of vehicle treated and gemfibrozil treated (A) WT, (B)PPARα^(−/−), & (C) PPARβ^(−/−) mice. Scale bar=20 μM. (D) Highermagnification images showing co localization of TPP1 and GFAP in thedentate gyrus of gemfibrozil treated mice (WT, PPARα^(−/−) andPPARβ^(−/−)). Scale bar=10 μM. (E-G) TPP1 levels were compared betweenthe CA1 region of vehicle treated and gemfibrozil treated (E) WT, (F)PPARα^(−/−) & (G) PPARβ^(−/−) mice. Scale bar=20 μM. (H) Highermagnification images showing co-localization of TPP1 and GFAP in the CA1region of gemfibrozil treated mice (WT, PPARα^(−/−) and PPARβ^(−/−)).Scale bar=10 μM. All results represent analysis of each of threehippocampal sections of each of four different mice per group.

FIG. 6 shows upregulation of TPP1 by fibrate drugs involves both PPARαand RXRα. (A-C) Mouse primary astrocytes were treated with differentconcentrations of all-trans retinoic acid (RA) and gemfibrozil and thecombination of the two in serum-free DMEM/F12 medium for 24 hrs followedby monitoring of mRNA expression of Cln2 by quantitative RT-PCR (A) andprotein expression of TPP1 by Western blot (B). (C) Denistometricanalysis of TPP1 (relative to β-actin) with RA and gemfibroziltreatment. p^(a)<0.05 vs WT control; p^(b)<0.05 vs 0.5 μM RA onlytreatment; p^(c)<0.05 vs 10 μM gemfibrozil only treatment. (D, E) Mouseprimary astrocytes were untransfected, transfected with scrambled siRNA(1.0 μg) or RXRα siRNA (1.0 μg) for 36 hrs followed by treatment with RA(0.5 μM) and gemfibrozil (10 μM) alone and in combination for 24 hrsserum free DMEM/F12 medium followed by RT-PCR for Rxrα, Rxrβ, Rxrγ (D)and quantitative real time PCR for Cln2 (E). p*<0.05 vs untransfectedcontrol; p**<0.05 vs scrambled siRNA transfected control; p^(d)<0.05 vsuntransfected-gemfibrozil treated sample; p^(e)<0.05 vs untransfected-RAtreated sample; p^(f)<0.05 vs scrambled siRNA transfected-gemfibroziltreated sample; p^(g)<0.05 vs scrambled siRNA transfected-RA treatedsample; ns—not significant w.r.t. RXR-α siRNA transfected control. (F)Mouse primary astrocytes were transfected with scrambled siRNA (1.0 μg)or RXRα siRNA (1.0 μg) and treated with gemfibrozil (10 μM) and RA (0.5μM) alone and in combination under similar culture conditions and theprotein expression of TPP1 were estimated by Western blot. (G)Denistometric analysis of TPP1 (relative to β-actin) with RA andgemfibrozil treatment. p**<0.05 vs scrambled siRNA transfected control;p^(h)<0.05 vs only gemfibrozil treatment; p^(i)<0.05 vs only RAtreatment; ns—not significant w.r.t. RXR-α siRNA transfected control.(H) Mouse primary astrocytes isolated from wild type, PPARα^(−/−) andPPARβ^(−/−) mice were treated with RA (0.5 μM) alone and in combinationwith gemfibrozil (10 μM) under similar culture conditions and cells weresubjected to Western blot for TPP1. (I) Densitometric analysis of TPP1levels (relative to β-Actin) in PPARα^(−/−) and PPARβ^(−/−) and wildtype astrocytes after RA and gemfibrozil+RA treatment. p^(†)<0.05 vs WTcontrol; p^(††)<0.05 vs PPARβ^(−/−) control; p^(j)<0.05 vs only RAtreatment in WT cells; p^(k)<0.05 vs only RA treatment in PPARβ^(−/−)cells; ns—not significant w.r.t. PPARα^(−/−) control. All results aremean±SEM of at least three independent experiments.

FIG. 7 shows fibrate drugs upregulating TPP1 via activation ofPPARα/RXRα heterodimer. (A) Mouse primary astrocytes were treated withgemfibrozil (10 μM) and RA (0.5 μM) alone and in combination in serumfree DMEM/F12 for 6 hrs and the nuclear extract was subjected to (i)immunoprecipitation by PPARα Ab followed by immunoblot for both RXRα andPPARα, (ii) immunoprecipitation by RXRα Ab followed by immunoblot forboth PPARα and RXRα, (iii) immunoprecipitation by control IgG followedby immunoblot for both PPARα and RXRα, (iv) nuclear extract wassubjected to immunoblot for PPARα, RXRα and Histone3 (H3). (B) Schematicdiagram for RXR binding site on the Cln2 promoter with the core sequenceand amplicon length. (C) Mouse astrocytes were treated with thecombination of gemfibrozil (10 μM) and RA (0.5 μM) for 6 hrs andrecruitment of PPARα and RXRα on the RXR binding site of Cln2 promoterwas monitored by ChIP analysis as described under “Materials andMethods”. Normal IgG was used as control. All results are representativeof at least there independent experiments.

FIG. 8 is a schematic representation of the mechanism of upregulation ofTPP1 by fibrate drugs via PPARα/RXRα pathway.

FIG. 9 shows the upregulation of lysosomal proliferation in primaryhuman astrocytes by gemfibrozil and fenofibrate. Cells were treated with25 μM gemfibrozil and 10 μM fenofibrate for 24 hrs followed by labelinglysosomes with LysoTracker. DAPI was used to visualize nuclei. Resultsrepresent 3 independent experiments.

FIG. 10 shows the upregulation of lysosomal proliferation in primaryhuman neurons by gemfibrozil and fenofibrate. Cells were treated with 25μM gemfibrozil and 10 μM fenofibrate for 24 hours followed by labelinglysosomes with LysoTracker. DAPI was used to visualize nuclei. Resultsrepresent 3 independent experiments.

FIG. 11 shows the upregulation of the lysosomal marker proteintripeptidyl peptidase 1 (TPP1) by gemfibrozil and fenofibrate. Cellswere treated with 25 μM gemfibrozil and 10 μM fenofibrate for 24 hoursfollowed by double-labeling for TPP1 and β-tubulin (neuronal marker).DAPI was used to visualize nuclei. Results represent 3 independentexperiments.

FIG. 12 shows the upregulation of lysosome-associated membrane protein 2(LAMP2) in human neurons by gemfibrozil and fenofibrate. Cells weretreated with 25 μM gemfibrozil and 10 μM fenofibrate for 24 hoursfollowed by double-labeling for LAMP2 and β-tubulin (neuronal marker).DAPI was used to visualize nuclei. Results represent 3 independentexperiments.

FIG. 13 shows the upregulation of lysosomal genes in brain cells bygemfibrozil and fenofibrate. (A) Quantitative real-time PCR forlysosomal genes in mouse astrocytes treated with 25 μM gemfibrozil fordifferent time points under serum free conditions. Results are mean±SDof three different experiments. ^(a)p<0.001 vs control. (B) RT-PCR forlysosomal genes in human astrocytes treated under same condition. (C)Human neurons were treated with different concentrations of fenofibratefor 24 hrs under serum free conditions and RT-PCR was done lysosomalgenes. Results represent 3 independent experiments.

FIG. 14 illustrates that gemfibrozil prolongs the life span of CLN2(−/−) mouse: CLN2(−/−) animals were orally administered with gemfibrozildissolved in 0.1% methyl-cellulose at a dosage of 7.5 mg/kg bodyweight/day. Treatment was started from 4 weeks of age for all groups.Methyl cellulose was used as vehicle. Top panel: Schematicrepresentation of experiments performed. Bottom panel: Kaplan-Meier plotfor percentage survival of treated vs. vehicle or untreated animals.

FIG. 15 illustrates that gemfibrozil treatment delays the loss of motoractivity in CLN2 (−/−) mice: Treated and untreated CLN2 (−/−) animalsfrom the previous experiment (FIG. 1) were monitored after 2 weeks (Leftpanel) and 6 weeks (right panel) of gemfibrozil treatment for variousmotor activity parameters. Each animal was allowed 5 mins of adaptationtime followed by monitoring for 5 min. *p-value <0.05. n=15 (untreated),n=20 (treated)

FIG. 16 illustrates that gemfibrozil treatment reduces peroxideproduction in CLN2 knockdown mouse astrocytes. Mouse astrocytes weretransfected with CLN2 siRNA (1.0 μg) for 36 hrs and treated withgemfibrozil (25 μM) for 6 hrs. Cells were then harvested and subjectedto peroxide assay. Results are mean±SD for three independentexperiments. * & **p-value<0.05

DETAILED DESCRIPTION

The present invention relates to methods of treatment of neuronal ceroidlipofuscinosis (NCL). Neuronal ceroid lipofuscinosis includes a group ofdiseases such as late infantile neuronal ceroid lipofuscinosis (LINCL)and Batten disease that are neurodegenerative lysosomal storagediseases. Many forms of NCL occur via mutations in the Cln genes. LINCLis associated with mutations in the Cln2 gene, which encodes tripeptidylpeptidase 1 (TPP1). The genes Cln1 and Cln3 are associated withinfantile NCL (INCL) and juvenile NCL (JNCL), respectively.

The methods of the present invention include administering to a subjectsuffering from NCL an agent that upregulates or enhances expression froma gene such as Cln1, Cln2, Cln3, and/or any combination thereof.Upregulation may include increasing mRNA levels for Cln1, Cln2, Cln3,and/or any combination thereof.

The methods of the present invention also include administering to asubject suffering from NCL an agent that upregulates TPP1 or restoresTPP1 activity. Upregulation may include increasing TPP1 mRNA levels,increasing TPP1 proteins levels, or increasing TPP1 enzymatic activity.The inventors have also discovered that activating a PPARα/RXRαheterodimer results in upregulation of TPP1. The inventors have alsosurprisingly shown that TPP1 is upregulated through the activity orinvolvement of PPARα, but not PPARβ or PPARγ.

The agent may be a lipid-lowering drug such as a fibrate. The fibratemay be gemfibrozil, fenofibrate, or clofibrate. Alternatively, the agentmay be all-trans retinoic acid. Surprisingly and unexpectedly,administration of the fibrate in combination with all-trans retinoicacid to the subject upregulates TPP1 more than administration of thefibrate or all-trans retinoic acid alone. The fibrate and all-transretinoic acid, when administered together to the subject, cooperativelyenhance upregulation of TPP1. In other words, a lower dose of thefibrate is needed in the presence of all-trans retinoic acid to achievethe same degree of TPP1 upregulation as occurs when only a higher doseof the fibrate is administered to the subject.

Both gemfibrozil and fenofibrate are capable of enhancing TPP1 incultured neurons and glial cells and in vivo in the brain. In addition,PPARα, but not PPARβ and PPARγ, is involved in gemfibrozil- andfenofibrate-mediated upregulation of TPP1. Furthermore, fibrate drugsupregulate TPP1 via activation of PPARα-RXRα heterodimer. Collectively,gemfibrozil and fenofibrate, FDA-approved drugs for hyperlipidemia, areof therapeutic value in the treatment of LINCL.

1. DEFINITIONS

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art. In case of conflict, the present document, includingdefinitions, will control. Preferred methods and materials are describedbelow, although methods and materials similar or equivalent to thosedescribed herein can be used in practice or testing of the presentinvention. All publications, patent applications, patents and otherreferences mentioned herein are incorporated by reference in theirentirety. The materials, methods, and examples disclosed herein areillustrative only and not intended to be limiting.

The terms “comprise(s),” “include(s),” “having,” “has,” “can,”“contain(s),” and variants thereof, as used herein, are intended to beopen-ended transitional phrases, terms, or words that do not precludethe possibility of additional acts or structures. The singular forms“a,” “and” and “the” include plural references unless the contextclearly dictates otherwise. The present disclosure also contemplatesother embodiments “comprising,” “consisting of” and “consistingessentially of,” the embodiments or elements presented herein, whetherexplicitly set forth or not.

For the recitation of numeric ranges herein, each intervening numberthere between with the same degree of precision is explicitlycontemplated. For example, for the range of 6-9, the numbers 7 and 8 arecontemplated in addition to 6 and 9, and for the range 6.0-7.0, thenumber 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 areexplicitly contemplated.

2. METHODS OF TREATING A NEURODEGENERATIVE DISEASE

Provided herein is a method of treating a neurodegenerative disease. Theneurodegenerative disease may be, for example, neuronal ceroidlipofuscinosis, Alzheimer's disease, Huntington's disease, Amyotrophiclateral sclerosis (ALS), Parkinson's disease, including Parkinson's plusdiseases such as multiple system atrophy (MSA), progressive supranuclearpalsy (PSP), corticobasal degeneration (CBD) or dementia with Lewybodies (DLB). The neurodegenerative disorder may be characterized bydefective autophage. Such disorders include Alzheimer's, Parkinson'sdisease, and Huntington's disease. The neurodegenerative disease may bea lysosomal storage disorder. The lysosomal storage disorder may be, forexample, Tay-Sach's disease, Fabry disease, Niemann-Pick disease,Gaucher disease, Hunter Syndrome, Alpha-mannosidosis,Aspartylglucosaminuria, Cholesteryl ester storage disease, ChronicHexosaminidase A Deficiency, Cystinosis, Danon disease, Farber disease,Fucosidosis, or Galactosialidosis.

The neuronal ceroid lipofuscinosis may be LINCL or Batten disease, forexample. The method may include administering to a subject sufferingfrom NCL an agent that upregulates TPP1 or restores TPP1 activity. Themethod may also include administering to a subject suffering from NCL anagent that upregulates or enhances expression from a gene such as Cln1,Cln2, Cln3, and/or any combination thereof.

NCL is a group of neurodegenerative diseases comprising typicalautosomal recessive lysosomal storage disorders. NCLs may includeclinical manifestations such as progressive mental deterioration,cognitive impairment, visual failure, seizures, and deteriorating motorfunction. NCLs may be associated with accumulation of autofluoroscentstorage materials in neurons and/or other types of cells. NCLs may bedivided into several types including Types 1 to 10, and the types may bebased on the age of onset, ultra structural variations in accumulatedstorage materials, and genetic alterations. Presently, no establishedtreatment and/or drugs are available for treatment of NCLs. Rather,present treatment is merely supportive of disease symptoms.

Many types of NCL may be associated with mutations in the Cln genes.These mutations may be associated with a deficiency or loss of function.For example, late infantile neuronal ceroid lipofuscinosis (LINCL) maybe associated with mutations in the gene Cln2, while infantile NCL(INCL) and juvenile (JNCL) may be associated with mutations in the genesCln1 and Cln3, respectively. The gene Cln2 encodes lysosomal tripeptidyltripeptidase 1 (TPP1), which is a 46 kilodalton protein that functionsin the acidic environment of the lysosomal compartment of a cell toremove tripeptides from the amino termini of proteins. Deficiency and/orloss of function of TPP1 may lead to intralysosomal accumulation ofautofluoroscent lipopigments known as ceroid-lipofuscins. Some subjectssuffering from NCL may have residual activity of TPP1.

a. Agent

The agent may be a lipid-lowering drug, all-trans retinoic acid, or acombination of the lipid-lowering drug and all-trans retinoic acid.Administration of the agent to a subject suffering from NCL mayupregulate or enhance expression from a gene such as Cln1, Cln2, Cln3,and/or any combination thereof. Upregulation may include increasing mRNAlevels for Cln1, Cln2, Cln3, and/or any combination thereof.

Administration of the agent to a subject suffering from NCL may alsoupregulate TPP1 or restore TPP1 activity. Upregulation of TPP1 mayinclude increasing the levels of TPP1 mRNA, increasing the levels ofTPP1 protein, or increasing the enzymatic activity of TPP1.Additionally, upregulation of TPP1 may include activating the PPARα/RXRαheterodimer, which is recruited to the promoter of the Cln2 gene toaffect TPP1 upregulation.

(1) Lipid-Lowering Drug

The agent mediating upregulation of TPP1 may be a lipid-lowering drug.Lipid-lowering drugs may be drugs that reduce the level of triglyceridescirculating in the blood of the subject. Additionally, lipid-loweringdrugs may be drugs that decrease the risk of hyperlipidemia. Suchlipid-lowering drugs may include fibrates such as gemfibrozil,fenofibrate, and clofibrate.

The fibrate may mediate upregulation of TPP1 via PPARα, but not PPARβand PPARγ. During upregulation of TPP1, PPARα forms a heterodimer withRXR-a and the RXR-α/PPAR-α heterodimer is recruited to the promoter ofthe Cln2 gene via a RXR binding site.

(2) All-Trans Retinoic Acid

The agent mediating upregulation of TPP1 may be all-trans retinoic acid.All-trans retinoic acid may also be known as ATRA, retinoic acid,tretinoin, and vitamin A acid. All-trans retinoic acid may mediateupregulation of TPP1 via the retinoid X receptor-α (RXR-α). Duringupregulation of TPP1, RXR-α forms a heterodimer with peroxisomeproliferator-activated receptor-α (PPAR-α) and the RXR-α/PPAR-αheterodimer is recruited to the promoter of the Cln2 gene via a RXRbinding site.

(3) Combination of Lipid-Lowering Drug and all-Trans Retinoic Acid

The agent mediating upregulation of TPP1 may comprise a combination ofthe lipid-lowering drug and all-trans retinoic acid. Such a combinationmay cooperatively mediate or enhance upregulation of TPP1 as compared toadministration of the lipid-lowering drug or all-trans retinoic acidalone. The combination may cooperatively enhance upregulation of TPP1about 2-fold, about 3-fold, about 4-fold, about 5-fold, or about 10-foldas compared to administration of the lipid-lowering drug or all-transretinoic acid alone. Particularly, the combination may cooperativelyenhance upregulation of TPP1 about 3-fold as compared to administrationof the lipid-lowering drug or all-trans retinoic acid alone.

b. Pharmaceutical Compositions

The agent may be incorporated into pharmaceutical compositions suitablefor administration to a subject (such as a patient, which may be a humanor non-human).

The pharmaceutical compositions may include a “therapeutically effectiveamount” or a “prophylactically effective amount” of the agent. A“therapeutically effective amount” refers to an amount effective, atdosages and for periods of time necessary, to achieve the desiredtherapeutic result. A therapeutically effective amount of thecomposition may be determined by a person skilled in the art and mayvary according to factors such as the disease state, age, sex, andweight of the individual, and the ability of the composition to elicit adesired response in the individual. A therapeutically effective amountis also one in which any toxic or detrimental effects of the agent areoutweighed by the therapeutically beneficial effects. A“prophylactically effective amount” refers to an amount effective, atdosages and for periods of time necessary, to achieve the desiredprophylactic result. Typically, since a prophylactic dose is used insubjects prior to or at an earlier stage of disease, theprophylactically effective amount will be less than the therapeuticallyeffective amount.

For example, a therapeutically effective amount of gemfibrozil may beabout 5 mg to about 2000 mg, about 10 mg to about 1900 mg, about 15 mgto about 1800 mg, about 20 mg to about 1700 mg, about 25 mg to about1600 mg, about 30 mg to about 1500 mg, about 35 mg to about 1400 mg,about 40 mg to about 1300 mg, about 45 mg to about 1200 mg, about 50 mgto about 1100 mg, about 55 mg to about 1000 mg, about 60 mg to about 900mg, about 65 mg to about 800 mg, about 70 mg to about 700 mg, about 75mg to about 600 mg, about 80 mg to about 500 mg, about 85 mg to about400 mg, about 90 mg to about 300 mg, about 95 mg to about 200 mg, orabout 100 mg to about 175 mg. In another example, the therapeuticallyeffective amount of gemfibrozil may be about 600 mg or about 1200 mg.

For example, a therapeutically effective amount of fenofibrate may beabout 5 mg to about 2000 mg, about 10 mg to about 1900 mg, about 15 mgto about 1800 mg, about 20 mg to about 1700 mg, about 25 mg to about1600 mg, about 30 mg to about 1500 mg, about 35 mg to about 1400 mg,about 40 mg to about 1300 mg, about 45 mg to about 1200 mg, about 50 mgto about 1100 mg, about 55 mg to about 1000 mg, about 60 mg to about 900mg, about 65 mg to about 800 mg, about 70 mg to about 700 mg, about 75mg to about 600 mg, about 80 mg to about 500 mg, about 85 mg to about400 mg, about 90 mg to about 300 mg, about 95 mg to about 200 mg, orabout 100 mg to about 175 mg. In another example, the therapeuticallyeffective amount of fenofibrate may be about 40 mg, about 48 mg, about54 mg, about 67 mg, about 100 mg, about 120 mg, about 134 mg, about 145mg, about 160 mg, or about 200 mg.

For example, a therapeutically effective amount of clofibrate may beabout 5 mg to about 2000 mg, about 10 mg to about 1900 mg, about 15 mgto about 1800 mg, about 20 mg to about 1700 mg, about 25 mg to about1600 mg, about 30 mg to about 1500 mg, about 35 mg to about 1400 mg,about 40 mg to about 1300 mg, about 45 mg to about 1200 mg, about 50 mgto about 1100 mg, about 55 mg to about 1000 mg, about 60 mg to about 900mg, about 65 mg to about 800 mg, about 70 mg to about 700 mg, about 75mg to about 600 mg, about 80 mg to about 500 mg, about 85 mg to about400 mg, about 90 mg to about 300 mg, about 95 mg to about 200 mg, orabout 100 mg to about 175 mg. In another example, the therapeuticallyeffective amount of clofibrate may be about 500 mg.

The pharmaceutical compositions may include pharmaceutically acceptablecarriers. The term “pharmaceutically acceptable carrier,” as usedherein, means a non-toxic, inert solid, semi-solid or liquid filler,diluent, encapsulating material or formulation auxiliary of any type.Some examples of materials which can serve as pharmaceuticallyacceptable carriers are sugars such as, but not limited to, lactose,glucose and sucrose; starches such as, but not limited to, corn starchand potato starch; cellulose and its derivatives such as, but notlimited to, sodium carboxymethyl cellulose, ethyl cellulose andcellulose acetate; powdered tragacanth; malt; gelatin; talc; excipientssuch as, but not limited to, cocoa butter and suppository waxes; oilssuch as, but not limited to, peanut oil, cottonseed oil, safflower oil,sesame oil, olive oil, corn oil and soybean oil; glycols; such aspropylene glycol; esters such as, but not limited to, ethyl oleate andethyl laurate; agar; buffering agents such as, but not limited to,magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-freewater; isotonic saline; Ringer's solution; ethyl alcohol, and phosphatebuffer solutions, as well as other non-toxic compatible lubricants suchas, but not limited to, sodium lauryl sulfate and magnesium stearate, aswell as coloring agents, releasing agents, coating agents, sweetening,flavoring and perfuming agents, preservatives and antioxidants can alsobe present in the composition, according to the judgment of theformulator.

c. Modes of Administration

Methods of treating neuronal ceroid lipofuscinosis may include anynumber of modes of administering the agent or pharmaceuticalcompositions of the agent. Modes of administration may include tablets,pills, dragees, hard and soft gel capsules, granules, pellets, aqueous,lipid, oily or other solutions, emulsions such as oil-in-wateremulsions, liposomes, aqueous or oily suspensions, syrups, elixiers,solid emulsions, solid dispersions or dispersible powders. For thepreparation of pharmaceutical compositions for oral administration, theagent may be admixed with commonly known and used adjuvants andexcipients such as for example, gum arabic, talcum, starch, sugars (suchas, e.g., mannitose, methyl cellulose, lactose), gelatin, surface-activeagents, magnesium stearate, aqueous or non-aqueous solvents, paraffinderivatives, cross-linking agents, dispersants, emulsifiers, lubricants,conserving agents, flavoring agents (e.g., ethereal oils), solubilityenhancers (e.g., benzyl benzoate or benzyl alcohol) or bioavailabilityenhancers (e.g. Gelucire™). In the pharmaceutical composition, the agentmay also be dispersed in a microparticle, e.g. a nanoparticulate,composition.

For parenteral administration, the agent or pharmaceutical compositionsof the agent can be dissolved or suspended in a physiologicallyacceptable diluent, such as, e.g., water, buffer, oils with or withoutsolubilizers, surface-active agents, dispersants or emulsifiers. As oilsfor example and without limitation, olive oil, peanut oil, cottonseedoil, soybean oil, castor oil and sesame oil may be used. More generallyspoken, for parenteral administration the agent or pharmaceuticalcompositions of the agent can be in the form of an aqueous, lipid, oilyor other kind of solution or suspension or even administered in the formof liposomes or nano-suspensions.

The term “parenterally,” as used herein, refers to modes ofadministration which include intravenous, intramuscular,intraperitoneal, intrasternal, subcutaneous and intraarticular injectionand infusion.

The present invention has multiple aspects, illustrated by the followingnon-limiting examples.

3. EXAMPLES Example 1 Materials and Methods for Examples 2-9

Reagents:

DMEM/F-12 50/50 1×, Hank's balanced salt solution (HBSS) and 0.05%trypsin were purchased from Mediatech (Washington, D.C.). Fetal bovineserum (FBS) was obtained from Atlas Biologicals (Fort Collins, Colo.).Antibiotic-antimycotic, gemfibrozil and Akt-inhibitor (Akt-i) wereobtained from Sigma-Aldrich (St. Louis, Mo.). Wortmannin and LY294002were purchased from Calbiochem (Darmstadt, Germany).

Isolation of Mouse Primary Astroglia:

Astroglia were isolated from mixed glial cultures as described (17, 18)according to the procedure of Giulian and Baker (19). Briefly, on day 9,the mixed glial cultures were washed three times with Dulbecco'smodified Eagle's medium/F-12 and subjected to shaking at 240 rpm for 2 hat 37° C. on a rotary shaker to remove microglia. After 2 days, theshaking was repeated for 24 h for the removal of oligodendroglia and toensure the complete removal of all nonastroglial cells. The attachedcells were seeded onto new plates for further studies.

Isolation of Primary Human Astroglia:

Primary human astroglia were prepared as described (20, 21). Allexperimental protocols were reviewed and approved by the InstitutionalReview Board of the Rush University Medical Center. Briefly, 11- to17-week-old fetal brains obtained from the Human Embryology Laboratory(University of Washington, Seattle, Wash., USA) were dissociated bytrituration and trypsinization. On 9th day, these mixed glial cultureswere placed on a rotary shaker at 240 rpm at 37° C. for 2 h to removeloosely attached microglia. On 11^(th) day, the flasks were shaken againat 190 rpm at 37° C. for 18 h to remove oligodendroglia. The attachedcells remaining were primarily astrocytes. These cells were trypsinizedand subcultured in complete media at 37° C. with 5% CO₂ in air to yieldmore viable and healthy cells. By immunofluorescence assay, thesecultures homogeneously expressed GFAP, a marker for astrocytes (22).

Isolation of Neurons from Different Brain Regions:

Fetal (E18-E16) mouse neurons were prepared as previously described (23)with modifications. Whole brains were removed and cortical, hippocampal,striatal and cerebellar fractions were dissected in serum freeNeurobasal media. The cells were washed by centrifugation three times at1200 rpm for 10 min, the pellet dissociated and the cells plated at 10%confluence in 8-well chamber slides pre-treated for >2 hr withPoly-D-Lysine (Sigma, St. Louis, Mo.). After 4 min, the non-adherentcell suspension was aspirated and 500 ml complete Neurobasal media(Invitrogen) supplemented with 2% B27 was added to each well. The cellswere incubated for 4 days prior to experimentation. Double-labelimmunofluorescence with β-tubulin and either GFAP or CD11b revealed thatneurons were more than 98% pure (data not shown). The cells werestimulated with gemfibrozil in Neurobasal media supplemented with 2% B27minus antioxidants (Invitrogen) for 24 hr prior to methanol fixation andimmunostaining.

Semi-Quantitative Reverse Transcriptase-Coupled Polymerase ChainReaction (RT-PCR):

Total RNA was isolated from mouse primary astrocytes and human primaryastrocytes using RNA-Easy Qiagen (Valencia, Calif.) kit followingmanufactures protocol. Semi-quantitative RT-PCR was carried out asdescribed earlier (24) using oligo (dT) 12-18 as primer and moloneymurine leukemia virus reverse transcriptase (MMLV-RT, Invitrogen) in a20 μl reaction mixture. The resulting cDNA was appropriately amplifiedusing Promega Master Mix (Madison, Wis.) and the following primers(Invitrogen) for murine genes:

(SEQ ID NO: 1) Mouse Cln1: Sense, 5′-ACACAGAGGACCGCCTGGGG-3′(SEQ ID NO: 2) Antisense, 5′-TCATGCACGGCCCACACAGC-3′ (SEQ ID NO: 3)Mouse Cln2: Sense, 5′-CACCATCCAGTTACTTCAATGC-3′ (SEQ ID NO: 4)Antisense, 5′-CTGACCCTCCACTTCTTCATTC-3′ (SEQ ID NO: 5)Mouse Cln3: Sense, 5′-TGCTGCCCTGCCATCGAGTG-3′ (SEQ ID NO: 6)Antisense, 5′-GGCAGCGCTCAGCATCACCA-3′ (SEQ ID NO: 7)Mouse Gapdh: Sense, 5′-GCACAGTCAAGGCCGAGAAT-3′ (SEQ ID NO: 8)Antisense, 5′-GCCTTCTCCATGGTGGTGAA-3′Amplified products were electrophoresed on 2% agarose (Invitrogen) gelsand visualized by ethidium bromide (Invitrogen) staining.Glyceraldehyde-3-phosphate dehydrogenase (Gapdh) mRNA was used as aloading control to ascertain that an equivalent amount of cDNA wassynthesized from each sample.

Quantitative Real-Time PCR:

The mRNA quantification was performed using the ABI-Prism7700 sequencedetection system (Applied Biosystems, Foster City, Calif.) using iTaq™Fast Supermix With ROX (Bio-Rad, Hercules, Calif.) and the following6-FAM/ZEN/IBFQ-labeled primers for murine genes: Cln2 and Gapdh(Integrated DNA Technologies Coralville, Iowa). The mRNA expression ofthe targeted genes was normalized to the level of Gapdh mRNA and datawas processed by the ABI Sequence Detection System 1.6 software.

Immunostaining of Cells:

Immunocytochemistry was performed as described earlier (25). Briefly, 8well chamber slides containing mouse primary astrocytes, mouse neurons,human primary astrocytes or SH-SY5Y cells were cultured to 70-80%confluence were fixed with chilled Methanol (Fisher Scientific, Waltham,Mass.) overnight, followed by two brief rinses with filtered PBS.Samples were blocked with 2% BSA (Fisher Scientific) in PBS containingTween 20 (Sigma) and Triton X-100 (Sigma) for 30 min and incubated atroom temperature under shaking conditions for 2 hr in PBS containing thefollowing anti-mouse primary antibodies: TPP1 (1:200; Santa CruzBiotech, Santa Cruz, Calif.), GFAP, (1:100; Santa Cruz), and β-tubulin(1:5000; Millipore). After four 15 min washes in filtered PBS, theslides were further incubated with Cy2 or Cy5-labeled secondaryantibodies (all 1:200; Jackson ImmunoResearch, West Grove, Pa.) for 1 hrunder similar shaking conditions. Following four 15 minute washes withfiltered PBS, cells were incubated for 4-5 min with4′,6-diamidino-2-phenylindole (DAPI, 1:10,000; Sigma). The samples wererun in an EtOH and Xylene (Fisher) gradient, mounted and observed underOlympus BX41 fluorescence microscope.

Immunostaining of Tissue Sections:

After 21 days of treatment, mice were sacrificed and their brains fixed,embedded, and processed. Sections were made from different brain regionsand for immunofluorescence staining on fresh frozen sections, anti-mouseTPP1 (1:200), goat anti mouse GFAP (1:100) were used. The samples weremounted and observed under Olympus BX41 fluorescence microscope (26).

Immunoblotting:

Western blotting was conducted as described earlier (27, 28) withmodifications. Briefly, cells were scraped in double-distilled H₂O andsodium dodecyl sulfate (SDS), electrophoresed on NuPAGE® Novex® 4-12%Bis-Tris gels (Invitrogen) and proteins transferred onto anitrocellulose membrane (Bio-Rad) using the Thermo-Pierce Fast Semi-DryBlotter. The membrane was then washed for 15 min in TBS plus Tween 20(TBST) and blocked for 1 hr in TBST containing BSA. Next, membranes wereincubated overnight at 4° C. under shaking conditions with the following1° antibodies; TPP1 (1:250, Santa Cruz), β-actin (1:800; Abcam,Cambridge, Mass. The next day, membranes were washed in TBST for 1 hr,incubated in 2° antibodies against 1° antibody hosts (all 1:10,000;Jackson ImmunoResearch) for 1 hr at room temperature, washed for onemore hour and visualized under the Odyssey® Infrared Imaging System(Li-COR, Lincoln, Nebr.).

TPP1 Activity Assay:

TPP-I activity was assayed in 96-well format plates using the followingmodification of the method described by Vines and Warburton (6).Briefly, samples and substrate (40 μL) were mixed in individual wells ofpolystyrene 96-well plate (Nalge Nunc International). The substratesolution consisted of 250 μmol/L Ala-Ala-Phe 7-amido-4-methylcoumarin(cat. no. A3401; Sigma; diluted freshly from a 25 mmol/L stock solutionin dimethyl sulfoxide stored at −20° C.) in 0.15 mol/L NaCl-1 g/L TritonX-100-0.1 mol/L sodium acetate, adjusted to pH 4.0 at 20° C. Plates werecentrifuged briefly to dispel bubbles and placed in a 37° C. Plates weremixed for 10 s before each reading. The plates were read from the bottomusing 360/20 nm excitation and 460/25 nm emission filters. Prior to theassay, the optimum substrate concentration and total protein in cellextract that can used to get best results were determined in the samemanner described above, using different substrate concentrations andprotein concentrations in the cell extract.

Immunoprecipitation from Nuclear Extract:

After treatment, cells were washed with PBS, scraped into 1.5 mL tubesand centrifuged in 4° C. for 5 min at 500 rpm. The supernatant wasaspirated and the pellet was resuspended in a membrane lysis buffercomprised of (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid)(HEPES, pH 8.0), MgCl₂, KCl, dithiothreitol (DTT) andprotease/phosphatase inhibitors, vortexed and centrifuged in 4° C. at15,000 rpm for 3 minutes. Again, the supernatant was aspirated and thepellet was resuspended in a high salt, nuclear envelope lysis buffercomprised of HEPES (pH 8.0), MgCl₂, glycerol, NaCl,ethylenediaminetetraacetic acid (EDTA), DTT and protease/phosphataseinhibitors, rotated vigorously in 4° C. for 30 min and centrifuged in 4°C. at 15,000 rpm for 15 minutes. The resultant nuclear pellet wasresuspended in IP buffer and a fraction was kept separately as lysate.The remaining nuclear extract was then precleared with 25 ul of proteinA-agarose (50%, v/v). The supernatants were immunoprecipitated with 5 ugof anti-RXRα or anti-PPARα or normal IgG (Santa Cruz Biotechnology,Inc.) overnight at 4 C, followed by incubation with protein A-agarosefor 4 hrs at 4° C. Protein A-agarose-antigen-antibody complexes werecollected by centrifugation at 12,000 rpm for 60 s at 4° C. The pelletswere washed five times with 1 ml of IP buffer (20 Mm Tris-HCl, pH 8.0,137 mM NaCl, 2 mM EDTA, 1% Nonidet P-40, 10% glycerol, 0.1 mMphenylmethylsulfonyl fluoride) for 20 min each time at 4° C. Boundproteins were resolved by SDS-PAGE, followed by Western blotting withthe anti-RXRα (1:2000, Santa Cruz) and/or anti-PPARα (1:250, SantaCruz). The lysate was resolved by SDS-PAGE followed by immunoblot forPPARα, RXRα and H3.

Chromatin Immunoprecipitation Assay:

ChIP assays were performed using method described by Nelson et al (29),with certain modifications. Briefly, mouse primary astrocytes werestimulated by 10 μM gemfibrozil and 0.5 μM RA together for 6 hrsfollowed by fixing with formaldehyde (1.42% final volume) and quenchingwith 125 mM Glycine. The cells were pelleted and lysed in IP buffercontaining 150 mM NaCl, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, NP-40 (0.5%vol/vol), Triton X-100 (1.0% vol/vol). For 500 ml, add 4.383 g NaCl, 25ml of 100 mM EDTA (pH 8.0), 25 ml of 1 M Tris-HCl (pH 7.5), 25 ml of 10%(vol/vol) NP-40 and 50 ml of 10% (vol/vol) Triton X-100 containing thefollowing inhibitors; 10 μg/ml leupeptin, 0.5 mM phenylmethlysulfonylfluoride (PMSF), 30 mM p-nitrophenyl phosphate, 10 mM NaF, 0.1 mMNa₃VO₄, 0.1 mM Na₂MoO₄ and 10 mM β-glycerophosphate. After one wash with1.0 ml IP buffer the pellet was resuspended in 1 ml IP buffer(containing all inhibitors) and sonicated and sheared chromatin wassplit into two fractions (one to be used as Input). The remainingfraction was incubated overnight under rotation at 4° C. with 5-7 μg ofanti-PPARα or anti-RXRα Abs or normal IgG (Santa Cruz) followed byincubation with Protein G-Agarose (Santa Cruz) for 2 hrs at 4° C. underrotation. Beads were then washed five times with cold IP buffer and atotal of 100 μl of 10% Chelex (10 g/100 ml H₂O) is added directly to thewashed protein G beads and vortexed. After 10 min boiling, theChelex/protein G bead suspension is allowed to cool to room temperature.Proteinase K (100 μg/nil) is then added and beads are incubated for 30min at 55° C. while shaking, followed by another round of boiling for 10min. Suspension is centrifuged and supernatant is collected. TheChelex/protein G beads fraction is vortexed with another 100 μl water,centrifuged again, and the first and the second supernatants arecombined. Eluate is used directly as a template in PCR. The followingprimers were used to amplify fragments flanking RXR binding element inthe mouse Cln2 promoter: Set1: sense: 5′-CAG CTG CCA TGT CCC CCA GC-3′,(SEQ ID NO: 9) antisense: 5′-TGC GCA GCT CTG TGT CAT CCG-3′ (SEQ ID NO:10); Set2: sense: 5′-GCT CCC TCT CCT CAG CTG CCA-3′ (SEQ ID NO: 11),antisense: 5′-CAT CCG GAG GCT CCA GGC CA-3′ (SEQ ID NO: 12). The PCRswere repeated by using varying cycle numbers and different amounts oftemplates to ensure that results were in the linear range of PCR.

Densitometric Analysis:

Protein blots were analyzed using ImageJ (NIH, Bethesda, Md.) and bandswere normalized to their respective β-actin loading controls. Data arerepresentative of the average fold change with respect to control forthree independent experiments.

Statistics:

Values are expressed as means±SEM of at least three independentexperiments. Statistical analyses for differences were performed viaStudent's T-test. This criterion for statistical significance wasp<0.05.

Example 2 Fibrate Drugs Upregulate TPP1 mRNA and Protein in MousePrimary Astrocytes

There have been reports that residual TPP-I activity can be found inpatients indicating that a few copies of normal Cln2 gene are left inpatients affected with LINCL (8, 9, 30, 31). We examined if FDA-approvedlipid-lowering drugs like gemfibrozil and fenofibrate were capable ofupregulating the expression of TPP1 in brain cells. Mouse primaryastrocytes were treated in serum free media with gemfibrozil withdifferent doses and for different time points. Both RT-PCR and real-timequantitative PCR (qPCR) analyses clearly indicated that gemfibrozilupregulated Cln2 mRNA levels in mouse primary astrocytes in a time anddose dependant manner with maximum increase at 24 hrs of 25 μMgemfibrozil treatment (FIGS. 1A, 1B, 1C & 1D). Other lysosomal geneslike Cln1 and Cln3 which are responsible for Infantile NCL (INCL) andJuvenile NCL (JNCL) respectively were also found to increase within 12to 24 h (FIG. 1A). The mRNA data was validated by Western blot where 3-4fold increase in TPP1 protein level was found with 25 μM gemfibrozil and10 μM fenofibrate treatment for 24 hrs. (FIGS. 1E and 1F)Immunofluorescence of primary mouse astrocytes stimulated withgemfibrozil and fenofibrate also revealed a dose dependant increase inTPP1 protein (FIG. 1G).

Example 3 Gemfibrozil and Fenofibrate Upregulate TPP1 in Neurons fromDifferent Parts of the Mouse Brain

Lack of TPP1 enzyme causes accumulation of lipofuscines in neuronsleading to loss of neurons in brain causing the disease to progress (5).Hence we examined the effect of the fibrate drugs in neurons anddetermined whether the induction of TPP1 occurs throughout the brain.Mouse primary neurons were isolated from different brain regions viz.cortex, hippocampus, and striatum and cultured and treated withgemfibrozil and fenofibrate. The immunofluorescence showed a significantincrease in the mouse neurons from all three brain regions (FIGS. 1H, 1J& 1L). Further, the neurons from those there brain regions were treatedwith gemfibrozil for 24 hrs followed by Western blotting for TPP1 whichshowed about 2-3 fold increase in TPP1 protein, as determined bydensitometric quantification (FIGS. 1I, 1K & 1M).

Example 4 TPP1 Proteins Upregulated by the Fibrate Drugs areFunctionally Active

Since the functional activity of the TPP1 protein is of criticalimportance in the clinical setting for LINCL (6), activity of the enzymewas measured. The cells were homogenized and the cell extracts weresubjected to TPP1 activity assay. Prior to that, the optimal substrateconcentration and optimal amount of extract for the assay was determinedby using different concentrations of substrate and sample respectively(Supplementary FIGS. 1A & 1B). TPP1 activity was measured (as describedin the methods section) in mouse primary neurons and mouse primaryastrocytes. The product formation increased with increasing doses oftreatment indicating an increase in activity of the protein in the cellextracts (FIGS. 1N & 1O). This can be attributed to the increase in thelevels of proteins in the cells observed in the earlier experiments.Collectively, these data strongly suggest that fibrate drugs can enhanceboth the mRNA and protein levels resulting in an increased activity ofthe protein in the cell.

Example 5 Fibrate Drugs Upregulate TPP1 mRNA and Protein in Human BrainCells

We further examined whether a similar increase in Cln2 mRNA and proteinwas obtained upon treatment of human cells with gemfibrozil andfenofibrate. Human astrocytes were treated in the same way as the mousecells and the mRNA levels were quantified. Again, both RT and qPCR dataindicated an increase in Cln2 mRNA levels in human astrocytes in a doseand time dependant manner with maximum at a dose of 25 uM gemfibrozil(˜15 fold) and at 12 hrs (˜10 fold) (FIGS. 2A, 2B, 2C & 2D). However,fenofibrate was seen to increase the mRNA levels at a relatively lowerdose (10 μM) but at same time point (12 hrs) as that of gemfibrozil(FIGS. 2E & 2F). Once again, the protein levels were assessed in humanastrocytes and SH-SY5Y cell lines by immunofluorescence and in both thecell types a considerable increase in the level of TPP1 protein wasobserved. (FIGS. 2G & 2H).

Example 6 PPARα is Involved in Fibrate Drug Mediated Upregulation ofTPP1

Since it is known that PPARs are activated by fibrate drugs, the role ofthese receptors in mediating upregulation of TPP1 protein was examined(15). Astrocytes isolated from PPARα^(−/−) and PPARβ^(−/−) and wild-type(WT) mice were treated with gemfibrozil and fenofibrate and Cln2 mRNAlevels measured. The data from semi-quantitative RT-PCR and qRT-PCRshowed that WT and PPARβ^(−/−) cells showed similar patterns ofupregulation, whereas PPARα^(−/−) cells showed little or no effect onthe upregulation of Cln2 mRNA expression upon gemfibrozil treatment(FIGS. 3A and 3B) and fenofibrate treatment (FIGS. 3C and 3D). Whenmouse primary astrocytes were treated with GW9662, a PPARγ antagonist,followed by gemfibrozil or fenofibrate treatment, there was increasedexpression of Cln2 mRNA, even in presence of the antagonist (FIGS. 3Gand 3H).

To confirm the mRNA measurements the WT and PPARα^(−/−) and PPARβ^(−/−)astrocytes were processed for protein analysis. The cells were treatedsimilarly with gemfibrozil and fenofibrate and immunofluorescence andWestern blotting were performed. The immunoblot and densitometricanalysis of the blots showed no significant increase in TPP1 levels inPPARα^(−/−) cells, but about 4-5 fold increase in WT and PPARβ^(−/−)cells (FIGS. 3E and 3F). The data from the Western blot was confirmed byimmunofluorescence where a similar effect of the drugs on WT and KOastrocytes was observed, i.e. little or no increase in TPP1 inPPARα^(−/−) cells, compared to WT and PPARβ^(−/−) cells (FIGS. 3I, 3J &3K).

Furthermore, the presence of active TPP1 enzyme was also confirmed bymeasurement of TPP1 activity in WT and KO cell types. The enzymaticactivity was drastically increased in WT and PPARβ^(−/−) cells upontreatment with gemfibrozil or fenofibrate (FIGS. 3L and 3N), whilePPARα^(−/−) cell extracts showed no significant increase in TPP1enzymatic activity (FIG. 3M). Collectively, these data indicate thatPPARα, but neither PPARβ nor PPARγ, is involved in the gemfibrozil- andfenofibrate-mediated upregulation of TPP1.

Example 7 TPP1 is Upregulated by Fibrate Drugs In Vivo in the CNS of WTand PPARβ^(−/−), but not in PPARα^(−/−), Mice

Once we confirmed the involvement of PPARα in the fibrate mediatedupregulation of TPP1 protein, we further checked whether the sameresults could be replicated in in vivo settings. WT, PPARα^(−/−) andPPARβ^(−/−) mice from same background were treated orally for 21 dayswith 7.5 mg/kg body wt/day gemfibrozil dissolved in 0.1%methylcellulose, which was also used as vehicle. At the end of thetreatment, the mice were killed and different regions of their brain,viz. Substantia nigra pars compacta, cortex, hippocampus, and dentategyrus were sectioned, and immunofluorescence was performed for thepresence of TPP1. Gemfibrozil treatment markedly increased the level ofTPP1 both in GFAP-positive cortical astrocytes (FIG. 4 A1, A2, A3 & A4)and NeuN-positive cortical neurons (FIG. 4 B1, B2, B3 & B4) in WT andPPARβ^(−/−), but not PPARα^(−/−), mice. Similarly, gemfibrozil treatmentalso increased the level of TPP1 in GFAP-positive astrocytes (FIG. 4 C1,C2, C3 & C4) and tyrosine hydroxylase-positive neurons (FIG. 4 D1, D2,D3 & D4) in the substantia nigra of WT and PPARβ^(−/−), but notPPARα^(−/−), mice. Gemfibrozil also increased TPP1 mostly in thenon-neuronal cells in the dentate gyrus (FIGS. 5A, 5B, 5C & 5D) and CA1region of the hippocampus (FIGS. 5E, 5F, 5G & 5H) of WT and PPARβ^(−/−),but not PPARα^(−/−), mice. These data clearly indicate that gemfibrozilincreases TPP1 in vivo in the CNS via PPARα.

Example 8 Upregulation of TPP1 by Fibrate Drugs Involve Both PPARα andRXRα

Next we investigated the mechanism of this upregulation. We observedthat Cln2 gene promoter lacked PPAR binding site but contained a RXRbinding site, instead. Due to the facts that RXRα is abundant in thebrain and astrocytes (32, 33) and that PPARα and RXRα form heterodimer,we thought the mechanism of upregulation of TPP1 may involveco-operative action of both PPARα and RXRα and not PPARα alone. Toverify our hypothesis, we performed an array of experiments. First, wechecked whether activating RXR by all-trans retinoic acid (RA), a knownactivator of RXRs, caused any change in the mRNA or protein levels ofTPP1. Interestingly, quantitative real time PCR data showed that even RAalone enhanced the mRNA levels of Cln2 (FIG. 6A). RA at a concentrationof 0.5 μM caused about 3.5 fold increase in Cln2 mRNA levels which iscomparable to the effect of 10 μM gemfibrozil treatment (˜4 fold) (FIG.6A). Moreover, when cells were treated with low dose of gemfibrozil (10μM) together with RA (at different concentrations), there was a profoundincrease in the Cln2 levels with optimum concentration of thecombination being at 10 μM gemfibrozil and 0.5 μM RA (about 12 foldincrease) (FIG. 6A). These mRNA data was validated by Western blotperformed in mouse astrocytes using the similar treatments (FIG. 6B).The densitometry analysis showed similar pattern of increase in theprotein levels of TPP1 as observed from the mRNA data (FIG. 6C). In bothreal-time PCR and immunoblot experiments the increase of TPP1 expressionwith the combinatorial treatment (10 μM gemfibrozil and 0.5 μM RA) wasfound to be statistically significant when compared to eithergemfibrozil (10 μM) or RA (0.5 μM) treatment alone. This finding clearlyindicates the possible involvement of RXR in the upregulation of theCln2 gene. Secondly, to confirm the involvement of RXRα, we knocked downRXRα in astrocytes by RXRα siRNA followed by treatment with gemfibroziland RA. The siRNA was found to specifically knockdown RXRα, but neitherRXRβ nor RXRγ (FIG. 6D). The effect of gemfibrozil and RA was found tobe abrogated in the absence of RXRα, as observed from the quantitativereal time PCR data (FIG. 6E). There was almost 8-10 fold increase in theCln2 level in both untransfected cells as well as cells transfected withscrambled siRNA, whereas cells with RXRα knockdown were almostunresponsive to the treatment of gemfibrozil or RA alone as well as thecombination (FIG. 6E). Similar results were obtained with the proteinanalysis. The denistometric analysis for the TPP1 Western blot showedalmost no enhancement of TPP1 levels in RXRα siRNA transfected cells(FIGS. 6F & 6G). Finally, to validate our hypothesis that both PPARα andRXRα are involved in the upregulation process, we checked whetheractivation of RXRα alone (in the absence of PPARα) can induce theexpression of Cln2. Mouse astrocytes from wild type (WT), PPARα^(−/−)and PPARβ^(−/−) mice were treated with RA (0.5 μM) and the combinationof gemfibrozil (10 μM) and RA (0.5 μM) followed by immunoblot analysisfor TPP1. It was observed that neither RA alone nor the combinationcould induce TPP1 in PPARα^(−/−) cells, whereas WT and PPARβ^(−/−) cellswere responsive to the treatment (about 5-6 fold induction of TPP1)(FIGS. 6H & 6I). These data suggest that either PPARα or RXRα alone isnot sufficient for the upregulation of TPP1.

Example 9 Fibrate Drugs Upregulate TPP1 Via Activation of PPARα/RXRαHeterodimer

After confirming the involvement of both PPARα and RXRα, we wereinterested to find out the actual role of the two factors. First, weexamined whether there was any actual physical interaction between PPARαand RXRα. Mouse astrocytes were treated with gemfibrozil and RAseparately as well as in combination and the nuclear extract wassubjected to Co-IP for both PPARα and RXR. Immunoprecipitation withPPARα Ab showed increased presence of RXR in the immunoblot for thetreated samples compared to control (FIG. 7A-i). Similarly, increasedabundance of PPARα was also observed when the nuclear extracts wereimmunoprecipitated with RXR Ab (FIG. 7A-ii). These results demonstratethe presence of PPARα/RXRα heterodimer in the nucleus of cellsstimulated with gemfibrozil and RA. These results are specific as we didnot find any bands with IgG (FIG. 7A-iii). Levels of PPARα and RXRα andHistone3 (H3) have been shown as loading controls (FIG. 7A-iv). Next, weperformed ChIP studies to show the recruitment of the PPARα and RXRα onthe RXR binding site on the Cln2 gene (FIG. 7B). Chromatin fragmentsfrom cells treated with gemfibrozil and RA were immunoprecipitated withboth PPARα Ab and RXRα Ab and the DNA obtained was amplified by PCR withprimers spanning the RXR binding site on the Cln2 gene promoter. In bothcases, we were able to amplify 200 bp fragments flanking the RXR bindingsite (FIG. 7C). In contrast, no amplification product was observed inany of the immunoprecipitates obtained with control IgG (FIG. 7C),suggesting the specificity of these interactions. These results suggestthat gemfibrozil and RA are capable of recruiting both PPARα and RXRα tothe RXR binding site of the Cln2 gene promoter (FIG. 7C).

Example 10 Summary of Examples 2-9

The classical late infantile neuronal ceroid lipofuscinosis (LINCLs) isan autosomal recessive disease, where the defective gene is Cln2,encoding tripeptidyl peptidase I (TPP1). At the molecular level LINCL iscaused by accumulation of autofluorescent storage materials in neuronsand other cell types. Currently there is no established treatment forthis fatal disease. This study reveals a novel use of gemfibrozil andfenofibrate, FDA-approved lipid-lowering drugs, in upregulating TPP1 inbrain cells. Both gemfibrozil and fenofibrate upregulated mRNA, proteinand enzymatic activity of TPP1 in primary mouse neurons and astrocytesas well as human astrocytes and neuronal cells. Since gemfibrozil andfenofibrate are known to activate peroxisome proliferator-activatedreceptor-α (PPARα), the role of PPARα in gemfibrozil- andfenofibrate-mediated upregulation of TPP1 was investigated revealingthat both drugs upregulated TPP1 mRNA, protein and enzymatic activityboth in vitro and in vivo in wild type (WT) and PPARβ^(−/−), but notPPARα−/−, mice. In an attempt to delineate the mechanism of TPP1upregulation, it was found that the effects of the fibrate drugs wereabrogated in the absence of retinoid X receptor-α (RXRα), a moleculeknown to form heterodimer with PPARα. Accordingly, all-trans retinoicacid, alone or together with gemfibrozil, upregulated TPP1.Co-immunoprecipitation and ChIP studies revealed the formation ofPPARα-RXRα heterodimer and binding of the heterodimer to RXR bindingsite on the Cln2 promoter. Together, this study demonstrates a uniquemechanism for the upregulation of TPP1 by fibrate drugs via PPARα: RXRαpathway.

Example 11 Discussion of Examples 2-9

The NCL family of disease can be considered to be one of the mostimportant hereditary neurodegenerative lysosomal storage diseases (LSD)in children (34). Mutations in the Cln2 gene result in deficiency orloss of function of the TPP1 enzyme (9, 30, 35). There have been reportsof over 68 missense mutations in the Cln2 gene including 35 single aminoacid substitutions. Studies with 14 different naturally occurringdisease associated mutations showed alteration of lysosomal transport,increased half-life of the proenzyme and improper folding, resulting toloss of function of the enzyme (9). Currently there is no establisheddrug mediated therapy for LINCL, a classic subtype of the NCLs. Studiesusing adeno-associated virus (AAV) and other viral vectors expressingrecombinant TPP1 demonstrate widespread expression of TPP1 and treatmentof Cln2-targeted mice with these recombinant vectors shows slowing ofdisease associated pathology and increase in survival in mutant mice(36-38). However, levels of TPPI activity achievable by AAV-mediatedgene therapy can vary and depend on various critical parameters andthere is considerable doubt whether similar effects can be achieved inhumans (36, 39)

On the other hand, restoration of activity at even low levels couldprove helpful for most LSDs, where restoration of even <10% of normalactivity may have therapeutic benefits (40). Studies with hypomorphs ofCln2 mutant mice, expressing different levels of TPP1 enzyme indicatethat even 3% of normal TPP1 activity is capable of delaying the onset ofthe disease and 6% of the normal activity attenuates the disease andincreases the lifespan of mice (40). Also two specific variants ofmutated TPP1 were responsive to molecular chaperone treatment,indicating that folding improvement strategies can be used to restorethe enzymatic activity (9). Recent studies suggest that some misfoldedvariants or misprocessed proteins may also be rescued by treatment inpermissive temperatures under suitable condition (9). There have alsobeen reports of some mutations in TPP1 (Arg447His), which apparently maynot have any pathogenic effect (31). Moreover, a sensitive enzymeactivity assay detected residual levels of TPP1 activity in variousbiological samples from patients who were confirmed to have LINCL bygenetic analysis (30). This study also showed the presence of enzymeactivity in various animals having NCL-like neurodegenerative symptomsrendering them unsuitable for being a model for classical LINCL (30).Furthermore, using highly sensitive capillary electrophoresis technique,Vigilio et al, reported that lymphocytes from patients affected withLINCL exhibited TPP1 activity, although at low levels (in a rangebetween 0.1 and 0.8 mU/mg) (8). These findings about the presence ofresidual enzymatic activity in LINCL patients are very interesting as itindicate the presence of at least a few copies of the functional gene inthe system. Therefore, identifying specific drugs and understanding themechanisms by which these drugs can upregulate the endogenous normalcopies of the gene may be a critical step for LINCL therapy.

Gemfibrozil, marketed as ‘Lopid’, and fenofibrate, known as ‘Tricor’,are FDA-approved drugs prescribed for hyperlipidemia (10, 12). Here wedelineate for the first time that, these drugs are capable ofupregulating TPP1 in brain cells. This finding was confirmed by bothmRNA and protein studies in both mouse and human cells. The increase inprotein levels was throughout the brain as neurons isolated fromdifferent brain regions of mouse showed increased TPP1 expression upontreatment with gemfibrozil. In case of LINCL, the presence offunctionally active TPP1 enzyme is critical for therapy, as we have torely on the upregulation of residual enzyme activity in patients. TheTPP1 activity assay, performed in different cell types, clearly showedthat there was significant increase in the activity of the enzyme whichis a result of increased levels of the protein. Considering thepossibility of treatment by upregulation of the endogenous Cln2 gene,this finding could be of importance in the therapy of LINCL.

Over the last few years, a number of studies emphasized the role ofPPARs in different regulatory and modulatory pathways. It is also wellknown that PPARα is activated by polyunsaturated fatty acids andoxidized derivatives and by lipid-modifying drugs of the fibrate family,including fenofibrate and gemfibrozil (41, 42). PPARα is present in thecytoplasm as an inactive complex with heat-shock protein 90 (HSP-90) andhepatitis virus B-X-associated protein-2 (XAP-2), which act as aninhibitor of PPARα. Fibrate drugs replace the HSP90 repressor complexand help to rescue the transcriptional activity of PPARα (15).Therefore, we investigated the role of the PPAR group of receptors inthis phenomenon. We examined all three PPARs, viz PPARα, PPARβ, PPARγfor their involvement in upregulation of TPP1. These studies clearlyindicate the involvement of PPARα, but not PPARβ and PPARγ, in thisprocess. In astrocytes from WT and PPARα^(−/−) and PPARβ^(−/−) mice,both the TPP1 mRNA and protein analysis showed the involvement of onlyPPARα. Involvement of PPARγ was ruled out as studies using knownantagonist of PPARγ revealed no effect. TPP1 enzyme activity in the cellextracts was also increased in WT and PPARβ^(−/−), but not PPARα^(−/−),cells. The in vitro studies were further validated by in vivo studies,where we used the knockout mice for PPARα and PPARβ. Our in vivo resultsalso supported the cell culture data.

In order to delineate the mechanism of fibrate drug-mediatedupregulation of TPP1, we analyzed the promoter region of the Cln2 gene.Surprisingly, no PPAR binding site was found in the mouse Cln2 promoter,but further analysis of the promoter revealed a RXR binding site. It iswell known that in order to bind to DNA and activate transcription, PPARrequires the formation of heterodimer with the retinoid X receptor (RXR)(43). Together the PPAR/RXR heterodimer regulates the transcription ofgenes for which products are involved in lipid homeostasis, cell growth,and differentiation (44, 45). This led us to think whether the pathwayof TPP1 upregulation requires a co-operative effect of both PPAR andRXR. It was observed that the activation of RXR by low doses of RA alone(0.5 μM) was capable of upregulating TPP1 to a comparable level of thatof gemfibrozil (10 μM). Also, when cells were treated with bothgemfibrozil and RA together, they co-operatively enhanced the expressionof TPP1 by almost more than 3 fold compared to the levels achieved byeither gemfibrozil or RA alone, which implies that a combinatorialtherapy could be more useful than using the compounds separately fortreatment. Furthermore, the effect of both gemfibrozil and RA wereabrogated in the absence of either RXRα or PPARα. The Co-IP studiesperformed with the nuclear extracts on astrocytes stimulated withgemfibrozil and RA demonstrated physical interaction between PPARα andRXR. These data clearly suggest that the treatment with gemfibrozil andRA activates both PPARα and RXRα which forms heterodimer in the nucleus.The ChIP data indicated the recruitment of the PPARα and RXRα on the RXRbinding site of the Cln2 promoter, hence validating our hypothesis.Collectively, these data outlines a unique mechanism where gemfibrozil,a known activator of PPARα, and RA, an agonist of RXRα, together canupregulate TPP1 in brain cells via PPARα/RXRα heterodimer.

Gemfibrozil and other fibrate drugs are known to reduce superoxide,lipid peroxidation products. It also strengthens the cellular defense bystimulating the activity of anti-oxidant proteins such as paraxonase andis associated with free radical scavenging ability as well as metal ionchelation. Therefore, apart from its lipid lowering effects, these drugsalso have anti-inflammatory, immunomodulatory and anti-oxidativeproperties (14, 27, 46-48). In the NCL cases, predominantly in LINCL,different brain regions have been shown to be immunoreactive for4-hydroxynonenal (4-FINE) or 8-hydroxydeoxyguanosine (8-OHdG), popularmarkers for evaluation of oxidative stress which may be caused due toaccumulation of lipofuscines and elevated cytokine response (1).Therefore, treatment with these drugs will not only lead to theupregulation of endogenous normal TPP1 leading to clearance oflipofuscines but also can be beneficial for the peripheral immune systemby downregulating the inflammatory pathways generated due toaccumulation of lipofuscines.

In summary, we have delineated that gemfibrozil and fenofibrate,FDA-approved lipid-lowering drugs, upregulate TPP1 in cultured mouse andhuman brain cells and in vivo in mouse brain via PPARα/RXRα pathway.Although the in vitro situation of mouse and human brain cells inculture and its treatment with gemfibrozil and fenofibrate may not trulyresemble the in vivo situation of the CNS of patients with lateinfantile neuronal ceroid lipofuscinosis (LINCLs), our results clearlyidentify these two drugs as possible therapeutic agents for LINCL thatcan be immediately taken to clinical trials for testing.

It is understood that the foregoing detailed description andaccompanying examples are merely illustrative and are not to be taken aslimitations upon the scope of the invention, which is defined solely bythe appended claims and their equivalents.

Various changes and modifications to the disclosed embodiments will beapparent to those skilled in the art. Such changes and modifications,including without limitation those relating to the chemical structures,substituents, derivatives, intermediates, syntheses, compositions,formulations, or methods of use of the invention, may be made withoutdeparting from the spirit and scope thereof.

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1. A method for treatment of a neurodegenerative disease, comprisingadministering to a subject in need of such treatment a compositioncomprising a therapeutically effective amount of an agent that mediatesupregulation of TPP1.
 2. The method of claim 1, wherein theneurodegenerative disease is selected from the group consisting ofneuronal ceroid lipofuscinosis, Alzheimer's disease, Huntington'sdisease, Amyotrophic lateral sclerosis (ALS), Parkinson's disease,including Parkinson's plus diseases such as multiple system atrophy(MSA), progressive supranuclear palsy (PSP), corticobasal degeneration(CBD) and dementia with Lewy bodies (DLB).
 3. The method of claim 1,wherein the neurodegenerative disease is a lysosomal storage disorder.4. The method of claim 3, wherein the lysosomal storage disorder isselected from the group consisting of Tay-Sach's disease, Fabry disease,Niemann-Pick disease, Gaucher disease, Hunter Syndrome,Alpha-mannosidosis, Aspartylglucosaminuria, Cholesteryl ester storagedisease, Chronic Hexosaminidase A Deficiency, Cystinosis, Danon disease,Farber disease, Fucosidosis, and Galactosialidosis.
 5. The method ofclaim 2, wherein the neurodegenerative disease is neuronal ceroidlipofuscinosis.
 6. The method of claim 1, wherein the agent is alipid-lowering drug.
 7. The method of claim 6, wherein thelipid-lowering drug is a fibrate.
 8. The method of claim 7, wherein thefibrate is gemfibrozil or fenofibrate.
 9. The method of claim 5, whereinthe neuronal ceroid lipofuscinosis is late infantile neuronal ceroidlipofuscinosis or Batten disease.
 10. The method of claim 1, whereinTPP1 is upregulated by increasing TPP1 mRNA levels, increasing TPP1protein levels, increasing TPP1 enzymatic activity, or activating aPPAR-RXR heterodimer.
 11. The method of claim 1, wherein the compositionfurther comprises a therapeutically effective amount of all-transretinoic acid.
 12. (canceled)
 13. The method of claim 11, whereinadministering all-trans retinoic acid and a fibrate provides a greatertherapeutic effect in the subject than administration of all-transretinoic acid or the fibrate alone.
 14. A method for treatment of aneurodegenerative disease, comprising administering to a subject in needof such treatment a composition comprising a therapeutically effectiveamount of an agent, wherein the agent restores TPP1 activity.
 15. Themethod of claim 14, wherein the neurodegenerative disease is selectedfrom the group consisting of neuronal ceroid lipofuscinosis, Alzheimer'sdisease, Huntington's disease, Amyotrophic lateral sclerosis (ALS),Parkinson's disease, including Parkinson's plus diseases such asmultiple system atrophy (MSA), progressive supranuclear palsy (PSP),corticobasal degeneration (CBD) and dementia with Lewy bodies (DLB). 16.The method of claim 14, wherein the neurodegenerative disease isneuronal ceroid lipofuscinosis.
 17. The method of claim 14, wherein theagent is a fibrate.
 18. The method of claim 17, wherein the fibrate isgemfibrozil or fenofibrate.
 19. The method of claim 17, wherein thetherapeutically effective amount of the fibrate is lower when thefibrate is administered in combination with all-trans retinoic acid. 20.A method for treatment of neurodegenerative disease, comprisingadministering to a subject in need of such treatment a compositioncomprising a therapeutically effective amount of an agent that mediatesupregulation of a gene selected from the group of Cln1, Cln2, Cln3, andany combination thereof.
 21. The method of claim 20, wherein theneurodegenerative disease is selected from the group consisting ofneuronal ceroid lipofuscinosis, Alzheimer's disease, Huntington'sdisease, Amyotrophic lateral sclerosis (ALS), Parkinson's disease,including Parkinson's plus diseases such as multiple system atrophy(MSA), progressive supranuclear palsy (PSP), corticobasal degeneration(CBD) and dementia with Lewy bodies (DLB). 22-24. (canceled)